Monoclonal antibody against group 2 allergen of dermatophagoides pteronyssiuns, hybridoma cell line producing thereof, strip, kit and method using said monoclonal antibody for dust mite assay

a monoclonal antibody and dermatophagoides technology, applied in the field of monoclonal antibody against der p 2, hybridoma cell line producing thereof, test strip, kit and method for dust mite assay, can solve the problems of allergenic type, antibody activity of wan-108 monoclonal antibody still remains elusive, and the effect of reducing the number of false positives

Inactive Publication Date: 2013-06-06
TAICHUNG VETERANS GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0025]Upon said technical characteristics above, this invention provides said kit and method for the use in dust mite assay by utilizing said monoclonal antibody against Der p 2 which performs immunological reaction with the environmental sample for precisely analyzing the presence and the number of dust mite allergen in the environment.

Problems solved by technology

However, there are still many uncertainties during the production of monoclonal antibody.
However, the allergenic type, recognized epitope and antibody activity of WAN-108 monoclonal antibody still remained elusive.

Method used

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  • Monoclonal antibody against group 2 allergen of dermatophagoides pteronyssiuns, hybridoma cell line producing thereof, strip, kit and method using said monoclonal antibody for dust mite assay
  • Monoclonal antibody against group 2 allergen of dermatophagoides pteronyssiuns, hybridoma cell line producing thereof, strip, kit and method using said monoclonal antibody for dust mite assay
  • Monoclonal antibody against group 2 allergen of dermatophagoides pteronyssiuns, hybridoma cell line producing thereof, strip, kit and method using said monoclonal antibody for dust mite assay

Examples

Experimental program
Comparison scheme
Effect test

example 1

Generation of Monoclonal Antibody Against Der p 2

Materials and Methods

[0040]1. Preparation of the Crude Extract of Dp

[0041]The dust mite is ground and suspended by PBS which contains protease inhibitor (aprotinin, 0.1 U / ml; Sigma Chemicals, St. Louis, Mo., USA) and PMSF (1 mmol / l, Sigma) for homogenization. After centrifuge at 10,000 g for 30 minutes, the supernatant is collected for dialysis in the 0.05 mol / l, pH 8.0 ammonium carbonate solution, and followed by aliquot and freeze dehydration. The concentration of the collected sample is estimated by Lowry's method with bovine serum as standard.

[0042]2. Production of Monoclonal Antibody Against Der p 2

[0043]The fusion protein of Der p 2 peptide fragments are expressed from the pGEX plasmid constructed by Dr. K. Y. Chua in Singapore University. The preparation procedure is examined as following: performing polymerase chain reaction (PCR) with different primer sets to obtain F1 to F16 fragments and then sub-cloning PCR products into g...

example 2

Generation of Dust Mite Test Strip

Materials and Methods

[0060]1. Determination of the Mixing Ratio of Gold Solution and Monoclonal Antibody C1

[0061]First, 500 μl of Gold solution (Evernew, Nano Gold-40, gold size 25-35 nm, Yu-Shing Biotech., Ltd., Taiwan) was divided into 9 tubes. Monoclonal antibody C1 was diluted with 0.005 mol / l, pH 9.0 borate buffer for preparing the diluted C1 antibody solution with the following concentrations: 0.1 μg / ml, 0.5 μg / ml, 0.75 μg / ml, 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 7.5 μg / ml and 10 μg / ml.

[0062]The aliquots of Gold solution are adjusted to pH 8.2 by using 0.1M HCl and 0.1M potassium carbonate solutions. The diluted C1 antibody solution is added into Gold solution and mixed for 5 minutes. Therein, one Gold solution without addition of diluted antibody solution was performed as negative control. All tubes were added with 500 μl of 10% NaCl and mixed well and then incubated for 2 hours. After incubation, the antibody / colloidal gold solution was analyzed by ...

experiment 3

Analysis of Dust Mite Number by Elisa Assay

Materials and Methods

[0075]1. Preparation of the Crude Extract of Dust Mite Protein

[0076]The protocol for crudely extraction of dust mite protein had been described in Experiment 1.

[0077]2. Preparation of rDer p 2 Protein

[0078]The rDer p 2 protein was produced by fermentation in 1000 ml conical flask. 200 ml BMGY (Invirtogen, USA) was added into conical flask for shaking at 30° C. with oxygen concentration at 35%. The medium adjusted to pH 7.0 by 15% ammonia was added with bacteria inoculum for fermentation. Until the glycerol was depleted, 50% glycerol was sequentially added into the fermentation medium; while the added glycerol was depleted, methanol was added to induce rDer p 2 expression with the loading rate according to pH and oxygen concentration. Finally, 1 ml of fermentation medium was subjected for characterization of rDer p 2 expression by using SDS-PAGE analysis.

[0079]3. Preparations of Monoclonal and Polyclonal Antibodies Again...

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Abstract

This present invention discloses a monoclonal antibody which specifically recognizes and binds to a epitope of group 2 allergen of Dermatophagoides pteronyssiuns, usually named Der p 2, and a hybridoma cell line producing thereof. Furthermore, this invention also discloses a strip, kit and method utilizing said monoclonal antibody for the detection of the presence of dust mite allergens and the calculation of dust mite number in the environment.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]A present invention discloses a monoclonal antibody against Der p 2, hybridoma cell line producing thereof and test strip, kit and method for dust mite assay.[0003]2. Description of the Related Art[0004]Asthma and allergic rhinitis are the most common airway allergic diseases in Taiwan. Both of them are resulted from the inflammation induced by the interaction between respiratory epithelial cells and allergens. The major allergens include hair of vertebrates, egg, murine urine, poisonous fluid of insect, dust mite, cockroach, shrimp, pollen, peanut and fungal. Therein, the dust mite exhibiting rich and diverse allergens majorly distributes at the food, furniture, interior decoration, bedding, carpet and dust within the indoor environment. Besides, allergens from dust mite are particularly easy to cause allergy of airway and skin. Therefore, dust mite is considered as the major allergen in the interior environment.[0005]...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/18G01N33/68
CPCC07K16/18G01N33/5308C07K2317/34G01N33/68
Inventor LIAO, EN-CHINTSAI, JAW-JI
Owner TAICHUNG VETERANS GENERAL HOSPITAL
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